Phosphorothioate RNAs are RNA analogues in which one of non-crosslinked oxygen atoms in the phosphodiester bond is replaced with a sulfur atom. Since phosphorothioate RNAs are expected to have higher nuclease resistance and cell membrane permeability compared with naturally occurring RNAs, they are considered to be most promising RNA-type nucleic acid medicaments at present (Mol. Cell., 6, pp. 1077-1087, 2000; Biochemistry, 42, pp. 7967-7975, 2003; Nucleic Acids Res., 31, pp. 589-595, 2003, and the like).
Phosphorothioate RNAs have a chiral center at a phosphorus atom, thereby two kinds of stereoisomers (Rp- and Sp-isomers) exist. It is known that these stereoisomers have different biochemical and physical properties (Proc. Natl. Acad. Sci. USA, 75, pp. 4798-4800, 1978; Biochemistry, 22, pp. 1369-1377, 1983). Therefore, when a phosphorothioate RNA is prepared, it is desired to selectively prepare a compound having a desired stereochemical configuration. However, it is difficult to prepare a phosphorothioate RNA under control of the steric configuration on the phosphorus atom, and accordingly, it has been desired to provide a method that can achieve efficient stereoselective synthesis.
As synthetic methods for phosphorothioate RNAs, there are known the enzymatic method (Nucleic Acids Res., 10, pp. 4145-4162, 1987), the H-phosphonate method (J. Org. Chem., 57, pp. 6163-6169, 1992), the H-phosphonate method combined with an enzymatic reaction (Nucleic Acids Res., 24, pp. 3811-3820, 1996), and the oxathiaphosphorane method (J. Org. Chem., 61, pp. 6713-6716, 1996). However, any of these methods are considered to be unsatisfactory from a viewpoint of stereoselectivity and the like.
As a method for synthesizing a phosphorothioate DNA, there is known the method in which an optically pure nucleoside 3′-oxazaphospholidine derivative derived from an optically active 1,2-amino alcohol as a monomer unit is coupled by using a weakly acidic activator having low nucleophilicity, N-(cyanomethyl)pyrrolidinium triflate (CMPT) (oxazaphospholidine method, J. Am. Chem. Soc., 130, pp. 16031-16037, 2008). In this method, use of bicyclic oxazaphospholidine provides marked difference in the activation energy between the diastereomers in the nucleophilic substitution reaction on a phosphorus atom, and thus high diastereoselectivity is attained.
Further, this method also comprises steps of condensing the monomer unit to the nucleoside derivative immobilized on the solid phase carrier such as controlled pore glass (CPG), and then capping unreacted 5′-hydroxyl group and the secondary amine of the asymmetric auxiliary group, followed by sulfurizing the phosphite, and removing 4,4′-dimethoxytrityl (DMTr) group at the 5′-end as deprotection. By repeating the steps according to an objective nucleotide sequence, a long chain phosphorothioate DNA can be synthesized. In the aforementioned publication, synthesis of a stereochemically pure thymidyl phosphorothioate decamer is reported.
There has been reported a method for stereoselectively synthesizing a phosphorothioate by applying the aforementioned oxazaphospholidine method to the synthesis of phosphorothioate RNA (Org. Lett., 11, pp. 967-970, 2009). By this method, a tetramer or higher oligomer was successfully synthesized by using tert-butyldimethylsilyl (TBDMS) group as a protective group of 2′-hydroxyl group of RNA, which is stable under the chain extension reaction conditions and can be easily removed with tetrabutylammonium fluoride (TBAF) under a neutral condition for deprotection, and by using CMPT as an activator. All-(Rp)-[Ups]9U and all-(Sp)-[Ups]9U are stereoselectively synthesized.
However, this method has problems of low reactivity and insufficient condensing efficiency for synthesis of a long chain oligomer. According to the researches by the inventors of the present invention, the condensing efficiency can be improved by using benzimidazolium triflate (BIT) or N-phenylimidazolium triflate (PhIMT) having nucleophilicity higher than that of CMPT as an activator, and uridyl phosphorothioate decamer can be stereoselectively synthesized. However, the use of the highly nucleophilic activator causes another problem that epimerization advances to reduce the diastereoselectivity.
As a protective group for 2′-hydroxyl group used in preparation of nucleic acid derivatives such as oligoribonucleic acids, cyanoethoxymethyl group (—CH2—O—CH2—CH2—CN, CEM) is known (Org. Lett., 7, pp. 3477-3480, 2005; International Patent Publication WO2006/22323). This protective group can be removed by reacting with fluorine ions under a neutral condition. An average condensing yield in coupling of monomers protected with TBDMS is 97% (Tetrahedron Letters, 43, pp. 795-797, 2002, page 795, left column, lines 8 to 11), whilst this protective group provides an average condensation yield of 99% or higher in a similar condensing reaction (Org. Lett., 7, pp. 3477-3480, 2005, page 3479, right column, lines 5 to 3 from the bottom). In the patent document disclosing the oxazaphospholidine method (WO2005/92909), 2-(cyanoethoxy)ethyl group (CEE) is exemplified as the protective group of the 2′-hydroxyl group. However, the document does not refer to cyanoethoxymethyl group.